A series of experiments was performed in 10- to 14-week old CD2F1 female mice, kept on a 12-hour light: 12-hour dark light regimen from birth. Time of day is referred to as Hours After Lights On (HALO). Tumors were established by transplantation, subcutaneously injected into the mouse flank with 5 × 105
viable meth-A sarcoma cells (provided by the laboratory of Dr. Lloyd Old). When tumor nodules were palpable, tumors were measured daily. When these tumors averaged 500 mm3
in volume, mice were either: (a) killed at one of six equispaced consecutive times of day (every 4 hours) to harvest tumor and host tissues, or (b) treated with intravenous 5-flourouracil (5-FU) chemotherapy drug at an effective dose at one of these same six times of day. Daily tumor measurements were continued and mice were followed until death [20
These tumor-bearing mice killed around the clock had bone marrow, intestinal epithelium, and tumor tissues removed, bisected, and stored at −80°C. The other half of bisected tumors were fixed in 10% buffered formalin for 24 hours and embedded in paraffin for subsequent staining and microscopic examination and immunohistochemical analysis. Thymidylate synthase (TS), an enzyme which, in the presence of folate and substrate, produces thymidylate, without which DNA cannot be synthesized, is the primary target of 5-FU antiproliferative effect. TS message, protein, and enzymatic activity were each measured in intestinal mucosa, bone marrow, and tumor samples obtained every 4 hours around a 24-hour period. Mitotic index, growth factor VEGF protein content, clock protein BMAL-1 nuclear and cytoplasmic content, and WEE1 protein (which gates mitosis) content were also measured in every tumor sample.
The tumor-bearing mice treated with 5-FU had measurements of daily serial tumor size, tumor disappearance rates, and survival assessments to determine therapeutic effects. 5-FU toxicity to healthy host cells was assessed by body weight fall and recovery, white blood cell fall and recovery, and perianal swelling (GI toxicity). A toxic-therapeutic index (TI) was then calculated for each 5-FU-treated mouse by combining the resulting drug toxicity to the host and tumor therapeutic efficacy scores. High TI values indicated a simultaneous occurrence of low host 5-FU toxicity with high anti-tumor therapeutic effects. Low TI values indicated high host 5-FU toxicity and low anti-tumor therapeutic effects.
These measurements allowed us to determine the circadian relationships among tumor growth, relevant tumor cell clock function, 5-FU target availability in normal and cancer tissues, and the circadian organization of the resultant 5-FU therapeutic index (anticancer efficacy and bone marrow and gut toxicity).
2.1. Multidimensional Processes
Movies were created in the form of 3-dimensional (3D) contour plots of three potential causally relevant factors at a time using bivariate interpolated values through Proc G3grid (SAS version 12, Cary, NC). The procedure creates a data set where the horizontal (x) and vertical (y) variable values form a complete grid, and it interpolates the value of the depth (z) variable for each point on the x-y plane. Assuming that the relationship between two times of day (HALO) measurements is linear, data obtained from two consecutive times of day (e.g., 2 and 6 HALO) were then linearly interpolated to create data that span about every 8 minutes between these two times of actual measurement.
In order to interpolate these data points, the following formula was used:
In Formula (1
refers to the value at a marked (measured) HALO point, H2
refers to the following data value at the next marked HALO point (4 hours later), n
refers to the number of time points to be interpolated, and P1
refers to the data value from the previous time point (8 minutes prior). For these sets of data, 30 was chosen as the number of data points, interpolated between each actual physical and biochemical measurement.
Upon completion of the linear interpolation method, the end result is a spreadsheet of values that span 121 rows and 181 columns of data values for each of these biologically relevant variables. We then use Co-Plot Software (Monterey, CA) to create 3D graphs, creating an 8-minute interval frame over a 24-hour span which is sequentially displayed to create movies depicting the 3D relationship over circadian time.
We have created three movies visualizing the relationships of an undulating surface changing over the 24-hour circadian cycle, comprised of the beating circadian clock as created from quantitative measurement of BMAL-1 clock protein in the tumor cell nucleus where it acts to keep time; the maximum amount of tumor shrinkage occurs as a result of circadian-timed 5-FU administration; the 5-FU Toxic-Therapeutic Index (TI) with (1) cell DNA replication, (2) cancer cell division and growth, and (3) 5-FU target, toxicity, and therapeutic effects. The undulating 3D surface relationships turn from blue (best), to purple (intermediate), to red (worst), to purple, to blue as TI changes rhythmically over circadian time. Because the rates of change of these relationships are also rhythmically changing throughout each day, a musical score is added in two acoustic dimensions. The melody cycles once throughout each completed circadian cycle. The beat/cadence of the musical score quickens as the changes in the relationships over these 24 hours accelerate and slows as they decelerate.
Finally, inspirational major chords accompany favorable circadian stages while melancholy minor chords accompany biomedically unfavorable relationships. Harmonically-favorable circadian stages are independently calculated by quantifying the survival duration of tumor-bearing mice treated at six circadian stages with 5-FU. This shape, color, and audio scheme concurrently depict the relationships among tumor cell circadian clock function, circadian tumor cell availability of the target of 5-FU, TS activity (TSA), the circadian therapeutic pattern of 5-FU induced tumor shrinkage, the TI of 5-FU treatment and the rate of change of these relationships within the day, and the overall survival benefit for tumor-bearing mice resulting from 5-FU treatment at six times of day.